To the Editor Schaefer et al. 1 (referred to as Study_1 ) recently presented the provocative conclusion that CRISPR-Cas9 nuclease can induce many unexpected off-target mutations across the genome that arise from the sites with poor homology to the gRNA. As Wilson et al. 2 pointed out, however, the selection of a co-housed mouse as the control is insufficient to attribute the observed mutation differences between the CRISPR-treated mice and control mice. Therefore, the causes of these mutations need to be further investigated. In 2015, Iyer et al. 3 (referred to as Study_2 ) used Cas9 and a pair of sgRNAs to mutate the Ar gene in vivo and off-target mutations were investigated by comparison the control mice and the offspring of the modified mice. After analyzing the whole genome sequencing (WGS) of the offspring and the control mice, they claimed that off-target mutations are rare from CRISPR-Cas9 engineering. Notably, their study only focused on indel off-target mutations. We re-analyzed the WGS data of these two studies and detected both single nucleotide variants (SNVs) and indel mutations.

Unexpected CRISPR off-target mutation pattern in vivo are not typically germline-like